| Objective: Atopic dermatitis(AD)is a highly prevalent skin disease in the world and one of the most common chronic inflammatory skin diseases.AD affects people of almost all ages and races,with prevalence rates reaching 3% in adults and 20% in children,and is increasing globally.AD is characterized by intense itching,and approximately 91% of AD patients suffer from chronic itching(lasting for more than 6weeks).As a result,patients’ sleep is seriously disturbed and their quality of life is severely impaired.In addition,AD ranks first in the global burden of disease caused by skin diseases.However,the pathogenesis of AD is not completely understood.The mechanisms are complex and diverse,involving strong genetic predispositions,epidermal dysfunction and multiple immune pathways dominated by T-cell inflammation.With regard to the treatment of AD,topical medication including corticosteroids,calcineurin inhibitors and phosphodiesterase inhibitors has been the mainstay of treatment for decades.However,long-term use may cause a series of side effects,such as skin atrophy caused by long-term use of corticosteroids and cancer risk induced by calcineurin inhibitors.Therefore,it is imperative to find new drugs with good curative effect and fewer side effects.For a long time,plants have played a vital role in the development of medicine and have been used to treat many different diseases in various ways.Arctii(F.arctii)is the mature fruit of Arctium lappa L.in the Compositae family,it has attracted great attention in folk medicine as a commonly used medicinal plant,and has been used in many fields such as treating fever,dizziness,diabetes,toothache,furuncle,and alopecia.Arctiin(ARC)has been listed as a marker compound and main active ingredient of Arctiin in Chinese pharmacopoeia,belonging to the lignan family.Arctiin is widely distributed,not only Arctium lappa contains a large amount of ARC,but also more than 38 other plants contain ARC.It has a variety of biological activities,including anti-inflammatory,anti-allergic,anti-cancer,anti-bacterial,anti-oxidation,anti-platelet activation,and can also be used as a calcium antagonist.Studies have shown that Arctium lappa extract can reduce the production of histamine and proinflammatory cytokines in mast cells and play an antiallergic role.ARC can alleviate IgE-mediated passive skin allergic reaction and anaphylactic shock induced by compound 48/80.At the same time,ARC has strong anti-inflammatory activity,such as inhibiting the transport pathway of nuclear factor(NF)-κB and the production of inflammatory mediators including IL-6,IL-1β and TNF-α.It can inhibit the activation of PI3K/AKT/NF-κB signaling pathway and improve acute lung injury in mice.In addition,ARC also ameliorated depression in mice by inhibiting microglia activation and inflammation through HMGB1/TLR4 and TNF-α/TNFR1 signaling pathways.However,the effect of ARC on atopic dermatitis is unclear.Therefore,the objective of our study is to evaluate the effects of ARC on atopic dermatitis of Balb/c mice induced by 2,4-dinitrochlorobenzene(DNCB)and human immortalized keratinocytes(HaCat cells)stimulated by TNF-α/IFN-γ.Methods: In this study,DNCB was first applied to the back skin of Balb/c mice to induce AD-like rash in mice to establish AD model.ARC was injected intraperitoneally for intervention,and Dexamethasone(DEX)group was set up as the positive control.The skin changes of mice were observed for dermatitis score,and the skin thickness of mice was measured at the same time.The histopathological changes of mice were evaluated by HE staining,the infiltration of mast cells in skin tissue of mice was evaluated by toluidine blue staining,and the changes of serum total IgE level in mice were detected by ELISA to determine the therapeutic effect of ARC on AD.At the same time,using western blot to detect the protein expressions of p-p65/p65、pIκBα/IκBα after ARC treatment,and the changes of mRNA levels of inflammatory factors IFN-γ and TSLP were detected by q-PCR.Then using western blot and immunohistochemistry to detect the expressions of TLR4,MyD88,IL-1β and NLRP3,respectively.The mRNA levels of apoptosis-related spot-like protein(ASC),Caspase-1 and IL-18 were detected by q-PCR to explore the mechanism of ARC on AD.In the cell experiment,CCK-8 was used to detect the cytotoxicity of ARC to HaCat cells.Then stimulate HaCat cells with TNF-α/IFN-γ to induce AD-like inflammatory reaction,and establish AD model in vitro.ARC was pretreated with different concentrations(100,200,400μg/m L)in experimental groups.The protein expressions of p-p65/p65、p-IκBα/IκBα,TNF-α and IL-6 were detected through western blot.The nuclear translocation of p65 in HaCat cells was observed by immunofluorescence,and the changes of cell microstructure were observed by electron microscopy to evaluate the anti-inflammatory effects of ARC on AD.Then the molecular docking of ARC and TLR4 was performed,and we detected the protein expressions of TLR4,MyD88,NLRP3,ASC,Caspase-1,IL-1β and IL-18 through western blot.The mRNA levels of TLR4,NLRP3 and IL-1β were detected using q-PCR,so as to further elucidate the mechanism of ARC on AD.Results:1.Regular application of DNCB induced scaly,scab and lichen-like changes in the skin of mice.HE staining of histopathologic sections showed epidermal thickening.The infiltration of mast cells was obvious in toluidine blue staining;Serum total IgE level increased.However,ARC treatment significantly alleviated the dermatitis of mice induced by DNCB,reduced the dermatitis score and the skin thickness,blocked the infiltration of mast cells,and decreased the serum IgE level,and the efficacy was comparable to DEX.Western blot results of skin tissue of mice in AD model group showed that the protein expressions of p-p65 and p-IκBα were remarkably enhanced,and the mRNA levels of IFN-γ and TSLP were notably increased detected by q-PCR.ARC treatment significantly inhibited the phosphorylation of p65 and IκBα,and decreased the mRNA levels of IFN-γ and TSLP,with no significant difference compared with DEX.2.The results of CCK-8 showed that ARC concentration of 400μg/m L had no effect on the cell viability of HaCat cells.TNF-α/IFN-γ stimulated HaCat cells to establish AD model in vitro,and the protein expressions of p-p65,p-IκBα,TNF-α and IL-6 were obviously enhanced,ARC pretreatment at different concentrations notably inhibited the phosphorylation of p65 and IκBα,decreased protein expressions of TNF-α and IL-6,and the effect was enhanced with the increase of concentration.At the same time,immunofluorescence showed that p65 was transferred from cytoplasm into the nucleus after TNF-α/IFN-γ stimulation of HaCat cells,and ARC preconditioning significantly blocked the nuclear translocation of p65.The results of electron microscopy showed that some of the nuclei of HaCat cells stimulated by TNF-α/IFN-γ were irregular,some were reduced in volume,most of the mitochondria were swollen,the mitochondrial ridge was discontinuous,the ridge structure disappeared,the ridge gap widened and vacuolated,and the vacuolation was observed.Autophagosome and autophagolysosome were significantly more than those in normal group.With ARC pretreatment,these microstructure changes have been obviously reduced.3.We carried out molecular docking between ARC and TLR4,and the results showed that ARC was combined in the binding pocket of TLR4,and seven hydrogen bonds were observed between them.Then,western blot was used to detect the protein expressions of TLR4,MyD88 and IL-1β,and immunohistochemistry was used to detect NLRP3 in the skin tissue of AD mice.The results showed that the protein expressions of them were greatly increased.q-PCR results showed that the mRNA levels of ASC,Caspase-1 and IL-18 were significantly enhanced.ARC treatment inhibited the enhancement of protein expression and mRNA level.Meanwhile,we also used western blot to detect the protein expressions of TLR4,MyD88,NLRP3,ASC、Caspase-1、IL-1β and IL-18 in TNF-α/IFN-γ stimulated HaCat cells,and the results showed that the protein expressions of them were significantly increased.The mRNA levels of TLR4,NLRP3 and IL-1β were obviously increased detected by q-PCR.ARC pretreatment with different concentrations suppressed the enhancement of protein expression and mRNA level,and the effect was enhanced as the concentration rised.Conclusion:1.ARC effectively alleviated AD-like dermatitis induced by DNCB in mice,reduced the thickness of skin,mast cell infiltration in skin tissue,and total IgE level in serum.In addition,ARC inhibited the activation of NF-κB pathway and transcription of Th1 and Th2 related cytokines,alleviating the symptoms of AD.2.ARC blocked the nuclear translocation of p65 in HaCat cells induced by TNF-α/IFN-γ,and decreased the protein expressions of TNF-α and IL-6 by inhibiting the activation of NF-κB pathway.At the same time,ARC also alleviated the inflammatory changes of HaCat cells induced by TNF-α/IFN-γ,inhibiting the inflammatory reaction of AD.3.ARC can combine with TLR4 and inhibit the activation of TLR4/MyD88/NF-κB/NLRP3 pathway in the treatment of AD. |
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